Most of what we know about antifungal drug research is derived from studies of non-pathogenic model fungi, such as Saccharomyces cerevisiae, which can easily be inserted into high throughput screening platforms, but are unsuitable as pan-fungal models. The rapid completion of genome sequences, and continually evolving bioinformatic manipulation of this vast and growing amount of data, has enabled new approaches to antifungal investigation. Presently, clinical mycology is faced with a growing problem of drug resistant and multi drug resistant organisms, which is compounded by the lack of approved antifungals and difficulty of discovering and bringing new ones to the clinic. These issues combine to limit antifungal choices for clinicians. To address this issue, we will develop a method that rapidly interrogates the fungal genome to reveal genes and pathways that are potential antifungal targets. The major objective of this study will be to develop a way to rapidly and inexpensively identify these targets. To accomplish this goal we will work with an increasingly important yeast pathogen, Candida glabrata, and an insertional mutagenesis system based on the bacterial pathogen, Agrobacterium tumefaciens. The first aim will be to improve the existing Agrobacterium tumefaciens transformation efficiency to yield enough transformants to produce a saturated insertional mutagenesis map. We will next develop a capture-probe based enrichment method for recovering insertion site fragments from the predominating non-junctional genomic DNA background. Finally, we will apply deep sequencing to these enriched fragments to identify each insertion site and its neighboring flanking genomic DNA, and ultimately assemble a high density insertion map that will be used to identify genes that are essential for survival, and therefore, potential antifungal targets. Three maps will be prepared, one will be a control map from the wild type genomic strain, and two will be maps prepared from cells exposed to Fluconazole and Caspofungin, which will be compared to the wild type map to identify drug-specific genes and genes that are responsive to both drugs. The long term goals that will be possible after this study will be to rapidly identify the genes and pathways that are the targets of any antifungal lead candidate.